Cells use ATP as energy currency to drive energy consuming cellular processes by linking them to the hydrolysis of ATP to ADP/AMP and phosphate. AMP-activated kinase (AMPK) senses the energy status in human cells. It becomes activated by the direct binding of AMP or ADP and is inhibited by ATP, which both compete for AMP/ADP binding. In addition, AMPK is inhibited by the glucose storage compound glycogen, which binds to a separate part of AMPK. Activated AMPK turns on ATP-generating pathways, such as glucose and fatty acid uptake and catabolism. It also turns down energy-consuming pathways, such as the synthesis of glycogen, fatty acids, cholesterol, rRNA, and proteins, as well as cell growth and proliferation. Due to its central roles in glucose metabolism and proliferation, AMPK is an important therapeutic target for the treatment of type 2 diabetes and cancer. AMP and ADP activate AMPK by changing AMPK's accessibility to upstream regulators (kinases and phosphatases). In addition, AMP and glycogen directly activate and inhibit the AMPK kinase activity, respectively, but the mechanism of this direct, allosteric regulation is unknown. Understanding how AMP and glycogen directly activate and inhibit AMPK requires high resolution crystal structures of AMPK in three relevant regulatory states (bound to AMP, bound to AMP and glycogen, and bound to ATP) to compare AMP- and ATP-bound states as well glycogen-bound and -free states. Obtaining these structures is hampered by unstructured regions that make AMPK recalcitrant to crystallization. We present an approach, validated by preliminary crystals and low resolution structures, to modify AMPK's crystal packing surfaces to allow the crystallization of AMPK in the presence of its unstructured internal regions and in complexes with its allosteric modulators. Comparison of these structures will allow us to identify changes in interactions and conformations associated with allosteric AMPK activation and inhibition. We will validate the analysis of our static crystal structures with extensive mapping o AMP- and cyclodextrin-induced local changes in surface accessibility by dynamic hydrogen/deuterium exchange mass spectrometry (HDX) and by mutational, biochemical, and cell-based analyses. Together, the results of the proposed studies will provide a detailed mechanism of the allosteric regulation of AMPK kinase activity and a structural basis to allow the rational design of novel AMPK modulators for the treatment of diabetes, obesity, and cancer.